Day 2
Day 2 Agenda
Goals
- Gain an overview of the challenges of transcript identification and the variety of computational methods.
- Gain practical proficiency in the identification of known and novel transcript isoforms, as well as quality control of transcriptomes using tools like
IsoToolsandSQANTI3. - Understand how to create structural annotations using an ab-initio predictor and further validate such annotations with long-read sequencing data.
- Gain practical experience in deriving isoform-resolved functional annotations and drawing biological conclusions through visualization and exploration.
Timetable
| Time | Activity | Details |
|---|---|---|
| 09:00 - 09:20 | Transcript Identification | Yalan & Fabian: Give an overview of basic concepts and the space of transcript identification and quantification tools (reference-reliant vs. de novo vs. quantification only) |
| 09:20 - 09:40 | IsoTools Theory | Yalan: Overview of theory and concepts of IsoTools algorithm and functionalities |
| 09:40 - 10:30 | IsoTools Practice | Yalan: Applying IsoTools to identify and quantify both known and novel transcripts, investigating results |
| 10:30 - 11:00 | Coffee Break | |
| 11:00 - 11:30 | SQANTI3 Theory | Fabian: Motivate why we need transcriptome quality control and filter, and introduce SQANTI3 concepts like structural categories |
| 11:30 - 12:20 | SQANTI3 Practice | Fabian: Applying SQANTI3 to perform quality control, filtering, and rescue on a custom transcriptome |
| 12:30 - 13:30 | Lunch break | |
| 13:30 - 14:00 | Structural Annotation Theory | Fabio: How long read sequencing integrates in the structural genome annotation workflow (ab initio predictions, exon vs intron merge) and why it is useful (identification of alternative splicing and lncRNA) |
| 14:00 - 14:30 | Structural Annotation Practice: Prediction | Fabio: Informing an ab-initio predictor with long-read models and evaluate its impact |
| 14:30 - 15:00 | Structural Annotation Practice: Validation | Fabio: Using long-read RNA support to validate gene models in a novel annotation |
| 15:00 - 15:30 | Coffee Break | |
| 15:30 - 16:00 | Functional Annotation Theory | Ana & Fabian: How to map functions (from public databases or predictors) to isoform-resolved sequences |
| 16:00 - 16:50 | Functional Annotation Practice | Ana & Fabian: Utilizing IsoAnnotLite to obtain isoform-resolved annotations and visualization of results |
| 16:50 - 17:00 | Summary | Questions |
Learning Objectives
- Students should be able to understand common problems for transcript identification and quantification and benchmarking resources
- Students should be able to understand the landscape of transcript identification and quantification tools.
- Students should be able to apply IsoTools and SQANTI3.
- Students should be able to understand how long read sequencing integrates in the structural genome annotation workflow.
- Students should be able to use long read sequences to validate gene models and assess performance
- Students should be able to functionally annotate transcript models and evaluate annotation results.
Materials
- (here we will put links to the slides)
Data
Recommended Bibliography
- Monzó, C., Liu, T. & Conesa, A. Transcriptomics in the era of long-read sequencing. Nature Reviews Genetics (2025). https://doi.org/10.1038/s41576-025-00828-z
- Bi, Yalan, et al. “IsoTools 2.0: software for comprehensive analysis of long-read transcriptome sequencing data.” Journal of Molecular Biology (2025): 169049. https://doi.org/10.1016/j.jmb.2025.169049
- Pardo-Palacios, F.J., Arzalluz-Luque, A., Kondratova, L. et al. SQANTI3: curation of long-read transcriptomes for accurate identification of known and novel isoforms. Nature Methods (2024). https://doi.org/10.1038/s41592-024-02229-2
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